Enhanced chemiluminescent reagent

Total proteins were extracted using Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China), and the proteins were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). Then, the membranes were placed in skim milk for blockage and probed with the primary antibodies against COMMD8 (K12416; 1:500; bjbalb, Beijing, China), Cyclin D1 (ab16663; 1:200; Abcam, Cambridge, MA. USA), Ki67 (ab16667; 1:1000; Abcam), B cell lymphoma/lewkmia-2 (Bcl-2) (ab185002; 1:1000; Abcam), Bcl-2 associated X Protein (Bax) (ab32503; 1:1000; Abcam), lactate dehydrogenase A (LDHA) (ab125683; 1:1000; Abcam), CD63 (ab134045; 1:1000; Abcam), CD81 (ab109201; 1:1000; Abcam) and β-actin (ab8227; 1:2000; Abcam) at 4°C for overnight. After the probe of the goat-anti-rabbit secondary antibodies on the next day, the protein signals were emerged using the enhanced chemiluminescent reagent (Beyotime) under an imaging system (Bio-Rad).

Colon tissue samples were acquired and lyzed with RIPA buffer (Beyotime Institute of Biotechnology). Protein was quantified using a BCA kit (Beyotime Institute of Biotechnology). A total of 100 µg protein per sample was boiled for 5 min prior to separation by 10% SDS-PAGE, and then transferred onto nitrocellulose membranes (Pall Life Sciences, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk at 37°C for 1 h and incubated with corresponding primary antibodies: IL-18 (cat. no. BS6823; 1:2,000; Bioworld Technology, Inc. St. Louis Park, MN, USA), phosphorylated (p-) signal transducer and activator of transcription (Stat) 3 (cat. no. 9145; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), total Stat3 (cat. no. 12640; 1:1,000; Cell Signaling Technology, Inc.) and GAPDH (cat. no. AP0063; 1:5,000; Bioworld Technology, Inc.) at 4°C overnight. Following 3 washes for 10 min in TBST, the membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h. Enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology) was used to develop the blots and protein was analyzed using Image Lab 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The colon tissues of the rats were lysed by radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China), and the concentration of the protein in each sample was determined by bicinchoninic acid kit (Beyotime Institute of Biotechnology). Then electrophoresis was performed, and the proteins on the gel were transferred onto polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA). Next, the membranes were blocked by 5% nonfat milk and incubated with the primary antibodies (anti-c-kit cat. ab32363, 1:1,000, anti-PAR-2 cat.ab138479, 1:1,000 and anti-GAPDH cat.ab9485, 1:2,000, all purchased from Abcam plc, Cambridge, UK) overnight at 4°C. On day 2, the membranes were washed and incubated with horseradish peroxidase conjugated secondary antibodies (cat.ab6721, 1:5,000, purchased from Abcam plc) at room temperature for 45 minutes. Finally, the membranes were washed again and incubated with the enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology). The signals were detected using Tanon 6100 Chemiluminescent Imaging System (Tanon Science & Technology Co., Ltd., Shanghai, China).

Total protein was extracted from rat hippocampal neurons using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China). Total protein was quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology) and 30 µg protein/lane was separated via SDS-PAGE on a 8% gel. The separated proteins were transferred onto polyvinylidene fluoride membranes and blocked for 1 h at room temperature with 5% non-fat milk. The membranes were incubated with primary antibodies against KLLN (1:1,000; cat. no. ab197892) and GAPDH (1:2,000; cat. no. ab9485; both Abcam, Cambridge, MA, USA) overnight at 4°C. Following primary incubation, membranes were incubated at room temperature for 45 min with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. ab6721; Abcam). Protein bands were visualized using the enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology) on a ChemiDoc XRS⁺ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein bands were quantified using ImageJ software (version 1.8, National Institutes of Health) with GAPDH as a loading control.

Protein was extracted from treated cells including Eca109 and KY-SE150 using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) with protease inhibitor (PMSF; Abcam) and quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (20 µg) were separated using 12% SDS-PAGE, and then transferred onto PVDF membranes. Subsequently, the membranes were incubated in 5% skimmed milk for 1 h at room temperature and then incubated with primary antibodies (Abcam), including SPATS2 (cat. no. ab122495; 1:500), cyclin E (cat. no. ab33911; 1:1,000), P53 (cat. no. ab32389; 1:1,000), matrix metalloproteinase (MMP-9; cat. no. ab73734; 1 µg/ml), N-cadherin (cat. no. ab202030; 1:500), and GAPDH (cat. no. ab181602; 1:10,000) at 4˚C overnight. After washing the membranes with TBS-Tween-20 (0.1%) three times, they were incubated with secondary antibody goat anti-rabbit IgG H&L (cat. no. ab205718; 1:2,000; Abcam) at room temperature for 1 h. After washing the membranes three times with TBS-T, the signals were detected using enhanced chemiluminescent reagent (Beyotime Institute of Biotechnology) and densitometry was estimated using Quantity One software (v4.6.6; Bio-Rad Laboratories, Inc.). GAPDH was used as a normalization control.